Molecular Immunology, Vol. 22, No. 8, lM?.~lOnS, 1985 Printed in Great Britain

0161~S89018S $3.W + O.@l 0 1985 Pergamon Press Ltd

PRELIMINARY COMMUNICATION

HUMAN CO~FLE~ENT &OMPONENT C3:

CHARACTERIZATION OF ACTIVE C3 S AND C3 F, THE TWO COMMON GENETIC VARIANTS

Niels Behrendt

Institute of Biochemical Genetics, University of Copenhagen, 2 A Oster Farimagsgade, DK-1353 Copenhagen K, Denmark

(Received 7 May 1985; accepted 31 May 1985)

Abstract.

The two common genetic variants of human C3, C3 S and C3 F, were purified and characterized by SDS-PAGE, aqarose gel electrophoresis, isoelectric fo- cusing and amino acid analysis. The difference in electrophoretic mobility between the two variants was conserved after purification, and by isoelectric focusing of the hemolytically active proteins, p1 values of 5.86 and 5.81 were determined for C3 S and C3 F, respectively. Any difference in amino acid com- position was too small to be detected by amino acid analysis, and the two proteins had the same molecular weight as determined by SOS-PAGE.

Introduction.

Human C3, the third component of complement, exhibits genetic polymorphism. The genetic variants of the protein are inherited as autosomal codominant traits. Several such variants have been found and characterized by agarose gel electrophoresis but only two, named C3 S (C3 slow) and C3 F (C3 fast), occur frequently. In most populations the sum of the gene frequencies of C3 S and C3 F is about 99 7;. The gene frequency of the most abundant variant, C3 S, ranges from 77 % - 99 % (Raum et al. (198O)).

Arvilommi (1974) has shown that the two variants differ in the capacity to bind to complement receptors on certain mononuclear cells in vitro. Further, the C3 type seems to be associated with certain diseases including rheumatoid arthritis (Bronnestam (1973)) and atherosclerotic disease (Sarensen & Dissing (1975)), but the mechanism that is responsible for this association is not known.

Several fragments of the C3 molecule display distinct roles in the immune system (reviewed by Brown et al. (1984)). Therefore, elucidation of the mole- cular difference between the two C3 variants will be important for the inter- pretation of any functional difference. The two variants have never been cha- racterized on the molecular level. This report describes the preliminary cha- racterization of the variants by SDS-PAGE, amino acid analysis and isoelectric focusing.

Materials and methods.

Fresh EDTA plasma from C3 S and C3 F homozygous donors was a kind gift from dr. H. Serensen, Blood Bank, State University Hospital (Rigshospitalet), Copenhagen. Rabbit anti C3c was from Dakopatts, Copenhagen. Standard proteins for SDS-PAGE (low molecular weight and high molecular weight calibration kits) and isoelectric focusing (pH 3-10 calibration kit) and ampholyte for isoelec- tric focusing (Pharmalyte 5-8) were from Pharmacia Fine Chemicals AB, Uppsala, Sweden.

C3 S and C3 F were purified from plasma pools derived from normal, healthy donors, homozygous for the given C3 type. Purification was performed according to Hammer et al. (1981) with minor modifications. The products were frozen in liquid nitrogen and kept at -7O'C. All analyses were performed on freshly thawn samples, and no sample was thawn more than once.

1006 NIELSBEHRENDT

The hemolytic C3 activity was measured by the AP C3 assay (Jessen et al. (1983)). Determination of the C3 type in plasma samples and purified prepa- rations was performed by agarose gel electrophoresis according to Teisberq (1970). Rocket and crossed immunoelectrophoresis were performed accordinq to Axelsen & Bock (1983) and Grubb (1983), respectively; 1D mM EDTA was included in the gel. SDS-PAGE was performed according to Laemmli (1970) (7.5 % slab gel). Pretreatment of samples for SDS-PAGE was performed at 37'C (Sim & Sim (1981)) to reduce autolytic cleavage of the o( chain of C3. Isoelectric focu- sing was performed in agarose gel, pH 5-8, following the recommendations of the manufacturer of ampholyte, except that the double ampholyte concentration was used. No detergents or denaturing agents were added to the gel. Prefocu- sing was omitted. The pH gradient was measured with a surface electrode at the temperature of focusing, i.e. lZ°C, and confirmed by comparison to p1 standard proteins. In some experiments, the focused proteins were allowed to diffuse into an alternative pathway C3 plate assay gel (Ploug et al. (1985)), instead of fixing and staining. Amino acid analysis was performed on a Waters amino acid analyzer. Analysis was performed under standard conditions, includinq separate determinations of cysteine (as cysteic acid) and tryptophan (after hydrolysis in 4 M methanesulfonic acid). For most amino acids, the reproduci- bility was better than 2.5 6.

Results.

Purification:

The two variants behaved identically during all purification steps except for DEAE Sephacel chromatography. When gradient elution was performed from a long (125 cm) column of this anion exchange resin, the elution volumes of C3 S and C3 F were slightly different, C3 5 eluting first. This was apparent in a similar purification experiment, in which C3 was purified from a plasma pool containing equal amounts of C3 S and C3 F. However, complete separation of the variants could not be obtained by this procedure.

Comparison of the antigenic C3 concentration (rocket immunoelectrophoresis) and hemolytic activity (AP C3 assay) in the raw material and purified prepa- rations showed that the hemolytic activity was fully retained after purifi- cation. Crossed immunoelectrophoresis of the purified variants against anti C3c showed one broad, symmetric precipitate similar to the C3 precipitate formed by fresh plasma samples.

Characterization of the purified variants:

C3 S and C3 F showed exactly the same pattern in SDS-PAGE. Reduced samples each showed two sharp bands corresponding to M values of 120,ODD and 78,000, respectively. No other bands were detected. SDS-PAGE of non-reduced samples showed an M r value of 200,000.

Agarose gel electrophoresis of the purified proteins showed that the dif- ference in electrophoretic mobility between C3 5 and C3 F was conserved. The electrophoretic mobilities were the same as in the C3 S and C3 F plasma sam- ples used as raw material. In each preparation one heavily staining band was observed. In addition, at a position slightly anodic to the main band, a faint band was detected. The same pattern was evident in the C3 region after elec- trophoresis of the raw material samples.

Isoelectric focusing of the purified proteins is shown in Fig. 1. The two proteins precipitated at the points of focusing, and the high concentration of ampholyte used (5.7 % w/v) was essential to avoid precipitation before focu- sing was completed. Each of the two preparations showed a heavily staining band and a faint, slightly more anodic band (lane 2 and 4). Each C3 variant focused at the same position whether applied anodic or cathodic to the point of focusing (lane 3 and 5). The isoelectric points corresponding to the heavi- ly staining bands were determined to be 5.86 and 5.81 for C3 S and C3 F, re- spectively. If, instead of fixing and staining, the focused proteins were al- lowed to diffuse into an alternative pathway C3 plate assay gel (Ploug et al. (1985)), hemolytic activity was observed at positions corresponding to the same pH values. The faint bands focused at positions corresponding to p1 values of 5.80 and 5.75 for the C3 5 and C3 F preparation, respectively.

The amino acid compositions of C3 S and C3 F, determined by amino acid ana- lysis of the purified variants, were identical within the reproducibility of analysis.

Human complementcomponentC3 1007

Fig. 1. Isoelectric focusing of purified C3 S and C3 F.

Focusing was performed at lZ°C for 2.5 hours at 10 W (3000 volt hours); inter electrode distance 10 cm. C3 samples con- tained 2 fig of protein. Bars to the left indicate the application positions relative to the anode (top of figure).

-cl) PI standard, applied at 8. (2) C3 F, applied at B. (3) Mixture of equal amounts of

C3 S and C3 F, applied at 8. (4) C3 S, applied at B. (5) Mixture of equal amounts of

C3 S and C3 F, applied at A (6) p1 standard, applied at A. p1 values of standard proteins are indicated to the right.

Discussion.

The two common human C3 variants, C3 S and C3 F, were purified to yield hemolytically active products. The purified variants were homogeneous accor- ding to SDS-PAGE and indistinguishable from the native proteins according to crossed immunoelectrophoresis.

Aparose gel electrophoresis and isoelectric focusing of the purified pro- teins showed that the charge difference between C3 S and C3 F is small, the p1 values differing by only 0.05 pH values. However, the difference is well de- fined and stable during purification.

Recently, it has been stated that human C3 exhibits an extensive charge heterogeneity with p1 values ranging from p1 5.5 - 6.5 (Vogel et al. (1984)). It was suggested that this heterogeneity, in addition to genetic polymorphism, reflected variations in the content of sialic acid. However, during investi- gation of the carbohydrate content of various human complement components, Tomana et al. (1985) failed to demonstrate any sialic acid in C3. In the pre- sent study, the genetic variants focused to well defined isoelectric points, provided a high concentration of ampholyte was used. If isoelectric focusing was performed at a lower concentration of ampholyte (2.9 % w/v), precipitation during focusing led to the appearance of protein containing zones covering a broad range of pH values.

As hemolytic activity could be demonstrated after isoelectric focusing, the p1 values presented here, p1 5.86 and 5.81 for C3 S and C3 F respectively, re- present the active proteins.

When the C3 type is determined in fresh plasma samples, a faint band is always observed at a position slightly anodic to the main C3 band (Teisberg (1970)). The same band was detected in the purified preparations after agarose gel electrophoresis or isoelectric focusing. The origin of this band is not known, but it is clearly connected to C3 as the distance to the main C3 band is the same in C3 S and C3 F homozygous plasma. The results presented here suggest that it is not a degradation product, as the M values determined by SDS-PAGE of reduced samples are in agreement with prevfously published values for the o( and p chains of C3, and no component of lower molecular weight could be detected.

The two variants had exactly the same electrophoretic mobilities in SDS- PAGE. This observation suggests that the polymorphism is caused by amino acid substitution or minor differences in post translational modifications, rather than the deletion of polypeptide segments or larger carbohydrate moieties.

No differences in amino acid composition could be detected by amino acid

analysis of C3 S and C3 F. The accuracy of analysis does not permit detection of single amino acid substitutions occurring among the more abundant residues. However, the result supports the conclusion that any difference in primary

1008 NIELS BE~~RENDT

structure must be limited to the substitution of one or very few amino acid residues.

Aknowledgements.

This work was supported by a grant from the Danish Rheumatism association. I wish to thank Dr. Henning Sorensen for providing the raw material for purifi- cation, Michael Ploug for carrying out the hemolytic plate assays, Drs. Vibeke Barkholt and Karen G. Welinder for valuable discussions and Solveig R. Jargen- sen for expert technical assistance.

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a property shared with ~~ macroglobu-

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